Characteristics of cardiopulmonary exercise testing in patients with combined post- and pre-capillary pulmonary hypertension due to left heart disease

Background Pulmonary hypertension (PH) is a common and morbid complication of left heart disease (LHD), comprising two subtypes: (1) isolated post-capillary pulmonary hypertension (Ipc-PH) and (2) combined post-capillary and pre-capillary pulmonary hypertension (Cpc-PH). Knowledge regarding the physiological characteristics that distinguish Cpc-PH, which has a worse prognosis, from Ipc-PH remains limited. Therefore, this study aimed to assess the utility of cardiopulmonary exercise testing (CPET) variables in detecting Cpc-PH. Methods and results Among 105 consecutive patients with LHD (age: 55 ± 13 years; male/female = 79/26) who underwent right heart catheterization and CPET, 45 (43%) were classified as PH-LHD (mean pulmonary artery pressure >20 mmHg). Ipc-PH (n = 24) was defined as pulmonary vascular resistance (PVR) ≤ 3 WU and Cpc-PH (n = 21) as PVR > 3 WU. Patients with Cpc-PH had a significantly lower peak partial pressure of carbon dioxide (PETCO2) (Non-PH/Ipc-PH/Cpc-PH = 38.2 ± 6.6 vs. 38.3 ± 6.0 vs 33.0 ± 4.4 mmHg, p = 0.006), higher VE vs. VCO2 slope (Non-PH/Ipc-PH/Cpc-PH = 33.0 [28.3, 36.6] vs. 32.5 [28.1, 37.8] vs. 40.6 [33.6, 46.1], p = 0.007), and lower ΔVO2/ΔWR (Non-PH/Ipc-PH/Cpc-PH = 8.5 ± 1.4 vs. 8.0 ± 1.7 vs. 6.8 ± 2.0 mL/min/watt, p = 0.001) than those with Ipc-PH and non-PH. Using multivariable logistic regression analysis, CPET variables were found to be independent predictors of Cpc-PH (lower peak PETCO2: odds ratio, 0.728 [95% confidence interval {CI}: 0.616–0.840], p = 0.003 and lower ΔVO2/ΔWR: odds ratio, 0.747 [95% CI: 0.575–0.872], p = 0.003). Conclusion From our exploratory analysis, CPET variables, especially in the lower peak PETCO2 and lower ΔVO2/ΔWR, were associated with Cpc-PH in patients with left heart disease.


Introduction
Pulmonary hypertension (PH) is a common and morbid complication of left heart disease (LHD). It comprises the following two subtypes: (1) isolated post-capillary PH (Ipc-PH) and (2) combined post-capillary and pre-capillary PH (Cpc-PH) [1]. Pathologically and genetically, Cpc-PH is considered to possess characteristics that are intermediate between those of pulmonary arterial hypertension (PAH), that is pre-capillary PH, and LHD, that is post-capillary PH [2][3][4]; furthermore, it has a worse prognosis than that of Ipc-PH. However, knowledge regarding the clinical or physiological characteristics that distinguish between these two sub-phenotypes remains limited [5]. The efficacy of pulmonary vasodilators in Cpc-PH has been explored recently [6,7]. Despite the increased importance of detection and differentiation of Cpc-PH to enable a tailored approach for PH treatment, an invasive evaluation for PH remains mandatory.
The cardiopulmonary exercise test (CPET) is a well-established noninvasive test for assessing functional capacity and exercise limitation. Based on pathophysiology, it provides mechanistic insights and important information on gas exchange, ventilatory efficacy, and cardiac function during exercise [8,9]. Peak oxygen consumption (peak VO 2 ), which reflects the cardiac output (CO) during exercise, and other ventilatory variables (such as the ventilation/carbon dioxide-production relationship slope [VE vs. VCO 2 slope]) remain the most frequently applied variables in the CPET and have been used as markers of disease severity and prognosis in patients with heart failure (HF) and PAH [10][11][12][13]. In terms of the CPET, PAH is characterized by a lower peak VO 2 , marked hyperventilation, a low end-tidal partial pressure of carbon dioxide (PETCO 2 ), and an elevated VE vs. VCO 2 slope [13,14]. The combination of a low PETCO 2 and high ventilatory equivalents for carbon dioxide (VE/VCO 2 ) at the anaerobic threshold (AT) has been considered suggestive of PH [15]. Previous reports on the differentiation of Cpc-PH have proposed the usefulness of various ventilatory parameters, such as the VE/VCO 2 at AT, PETCO 2 , dead-space ventilation-to-tidal ventilation (VD/VT), and lowest VE/VCO 2 %pred; however, no consensus has been reached yet [16,17].
An examination of the CPET variables in patients with Cpc-PH may provide mechanistic insights into the pathophysiology of Cpc-PH and facilitate the detection of early abnormal adaptations leading to Cpc-PH. Therefore, this study aimed to assess the utility of CPET variables in the non-invasive detection of Cpc-PH, which is considered to possess characteristics of both pre-capillary and post-capillary components.

Methods
This study complied with the principles of the Declaration of Helsinki and was approved by the Committee for Clinical Studies and Ethics of the Kyorin University School of Medicine (No 1261). Written informed consent was waived by the ethics committee because of the retrospective nature of this study.

Study patients
We retrospectively enrolled patients with LHD undergoing both right heart catheterization (RHC) and CPET under stable clinical conditions at our hospital between 2012 and 2017. During this period, elective RHC was performed in 255 patients whose primary diagnosis was LHD. Patients whose primary diagnosis was not LHD (e.g., right-sided HF, constrictive pericarditis, high-output HF) and those whose RHC was performed in urgent settings (e.g., acute coronary syndrome, cardiogenic shock) were not included. The clinical indication for RHC was according to the international guideline [18]. In total, 2,211 patients, without comorbidities influencing exercise performance or usual contraindications for CPET, underwent CPET for the purpose of exercise prescription or exercise tolerance assessment. Finally, the study population comprised 105 consecutive hospitalizedpatients with LHD who underwent both RHC and CPET. Patients with dilated cardiomyopathy (n = 56), hypertrophic cardiomyopathy (n = 3), secondary cardiomyopathy (n = 6), ischemic cardiomyopathy (n = 13), valvular heart disease (n = 13), hypertensive heart disease (n = 5), and diastolic dysfunction (n = 9) were included.
The baseline hemodynamic data were recorded; the zero-reference level (mid-chest) was adjusted at the commencement of pressure measurement, and the pulmonary artery wedge pressure (PAWP) was obtained as the mean value of the arterial trace during occlusion. Measurements were obtained at the end of a normal expiration with the patients in a resting-state supine position to assess the right chamber, right atrium pressure (RAP), right ventricular end-diastolic pressure, pulmonary artery pressure (PAP; mean PAP, systolic PAP, and diastolic PAP), and PAWP [19]. The O 2 saturation in the pulmonary artery (SvO 2 ) and in the arterial blood i.e. in the radial or femoral artery (SaO 2 ) was measured. The CO was determined by the Fick method using the following formula: CO (L/min) = VO 2 /(1.34 × hemoglobin × [SaO 2 -SvO 2 ]). The pulmonary vascular resistance (PVR) was calculated as follows: PVR (Wood units [WU]) = (mean PAP-PAWP)/CO. The diastolic pulmonary pressure gradient (DPG) was calculated as follows: DPG = diastolic PAP-PAWP. The pulmonary arterial compliance (PAC) were calculated as: PAC (ml/mmHg) = stroke volume/ pulse pressure.

Hemodynamic definition
To investigate the hemodynamics according to the presence of pulmonary vasculopathy, we divided patients into the following PH subgroups according to the recommendations in 2019 [1]: (i) non-PH group (mean PAP � 20 mmHg), (ii) Ipc-PH group (mean PAP >20 mmHg with PVR � 3.0 WU), and (iii) Cpc-PH group (mean PAP >20 mmHg with PVR >3.0 WU).

CPET
An incremental symptom-limited exercise test was performed within 3 weeks of RHC using an electromagnetically braked cycle ergometer (Strength Ergo 8, Fukuda Denshi, Tokyo, Japan) according to the ramp protocol. The test comprised a 3-minute resting period, followed by 3 min of warm up at an ergometer setting of 10 W (60 rpm); this was subsequently followed by testing that involved a 1-2 W increase in the exercise load every 6 s (10-20 W/min), depending on the predicted maximum exercise capacity, such that a maximal effort was attained within 8-15 min. The heart rate, arterial blood pressure in the brachial artery, and electrocardiogram were monitored continuously during the test.
During exercise, oxygen consumption (VO 2 ), carbon dioxide output (VCO 2 ), and minute ventilation (VE) were measured using a metabolic cart (AE-302S; MINATO, Tokyo, Japan). Prior to calculating the parameters from the respiratory gas analysis, an eight-point moving average of the breath-by-breath data was obtained. Peak VO 2 was defined as the average value obtained during the last 30 s. The AT point was determined using the V-slope method in addition to the following conventional criteria: VE/VO 2 increased after registering as flat or decreasing, whereas VE/VCO 2 remained constant or decreased [20,21]. The VE vs. VCO 2 slope was calculated from the commencement of incremental exercise to the respiratory compensation (RC) point using least squares linear regression [11]. The PETCO 2 was recorded at rest, AT, and peak exercise. The slope of VO 2 increase to work-rate increase (ΔVO 2 /ΔWR), reflecting the rate of CO increase, was calculated from the data recorded between 30 s after the commencement of incremental exercise and 30 s before the end of the exercise using least squares linear regression.

Echocardiography
Transthoracic Doppler echocardiography was performed, and echocardiograms were stored digitally on an ATRADA (Cannon, Japan) ultrasound system. The frame rate was maintained at a minimum of 60/s. For Doppler recordings, an average of 3-5 consecutive beats were measured using a horizontal sweep of 75-100 cm/s. The left ventricular (LV) dimensions and left atrial diameter (LAD) were measured from the parasternal long axis view. The LV ejection fraction (LVEF) was calculated using Simpson's biplane method from the apical four-and two-chamber views. Mitral inflow was assessed in the apical four-chamber view with the pulsed-wave Doppler sample volume placed at the tips of the mitral valve leaflets during diastole; the early (E) and late (A) peak diastolic velocities of mitral inflow and the E wave deceleration time were thus measured. Mitral annular motion was assessed using pulsed-wave tissue Doppler with the sample volume placed in the septal (e' septal). The E/e ratio was calculated.

Statistical analysis
Analyses were performed using SPSS (version 26.0; IBM Corp., Armonk, NY, USA). The Shapiro-Wilk test and histogram analyses were performed to assess for normality. Continuous variables are presented as mean ± standard deviation or median (25 th , 75 th interquartile range), where appropriate. Comparisons of more than two groups were performed using a one-way analysis of variance (with the Bonferroni post-hoc test) or the Kruskal-Wallis test (with the Dunn's post-hoc test), where appropriate. Categorical variables are presented as percentages and were compared using the Fisher's exact test or the Pearson's χ 2 test. Univariable and multivariable logistic regression analyses were performed to predict Cpc-PH using the CPET parameters. Significant independent variables were identified using stepwise selection (p<0.05). Receiver operating characteristic curves were constructed, and the area under the curve (AUC) was calculated. The cutoff value resulting in the highest product of sensitivity and specificity was considered optimal for the detection of Cpc-PH. The relationship between hemodynamic phenotype and all-cause death was evaluated with Kaplan-Meier analysis. Differences between survival curves were assessed using the log-rank test. The association between the CPET parameters and mortality was assessed using Cox proportional hazard models. Variables that were significant in the univariate analysis were entered into the multivariate Cox proportional hazard models. Statistical significance was set at p <0.05.

General characteristics
The 105 enrolled patients with LHD who underwent RHC and the CPET were predominantly male (75%), with a mean age of 55 ± 13 years and a mean LVEF of 39 ± 14%. Overall, 45 patients (43%) were classified as having PH-LHD. Among them, 24 patients (23%) had Ipc-PH and 21 patients (20%) had Cpc-PH (Fig 1). The general characteristics of the non-PH, Ipc-PH, and Cpc-PH groups are shown in Table 1. There were no significant differences in the age, LHD etiology, and hemoglobin level among the three groups. Patients in the Cpc-PH group were predominantly male, had higher brain natriuretic peptide levels, and had lower LVEF as compared to the levels of those in the non-PH and Ipc-PH groups.

Hemodynamic parameters
The hemodynamic parameters of the study groups are shown in Table 1. The PAWP, PAP, and mean RAP were significantly higher in the Ipc-PH and Cpc-PH groups than in the non-PH group. However, these variables did not differ between the Ipc-PH and Cpc-PH groups. The CO was significantly lower in the Cpc-PH group than in the non-PH and Ipc-PH groups (non-PH vs. Ipc-PH vs. Cpc-PH = 3.8 [

Discussion
In our exploratory analysis, we investigated the CPET parameters in patients with non-PH, Ipc-PH, and Cpc-PH according to the criteria of 2019 [1]. In our cohort, 35% of the patients who underwent RHC had PH-LHD, with Cpc-PH accounting for 20% of the entire cohort. Therefore, Cpc-PH appeared to be a relatively uncommon condition, consistent with previous findings [17]. The present study's results also revealed that the peak effort PETCO 2 and ΔVO 2 /ΔWR were the optimal predictors of Cpc-PH, thereby corroborating the findings of previous studies where ventilatory variables from CPET proved useful in differentiating Cpc-PH [16,17].

Characteristics of Cpc-PH
PH is a common complication of LHD, and it develops in response to a passive increase in left-sided filling pressures, more specifically the left atrial pressure. While it is associated with a poor prognosis [22], Cpc-PH is known to have an even worse prognosis [5].
Genetically, Cpc-PH resembles PAH. Assad et al. found that patients with Cpc-PH had genetic abnormalities in pathways that were highly active in the lungs and were known to contribute to the pathophysiology of PAH. These exploratory genetic findings suggest that Cpc-PH may have a pathophysiology distinct from that of Ipc-PH [2].
From a pathological perspective, progressive thickening and collagen proliferation occur in the lamina densa in order to protect against fluid accumulation in the interstitium of the endothelium and the vascular wall as well as in the alveoli [3,4,23]. In Ipc-PH, small arteries exhibit endothelial dysfunction and vasoconstriction, despite no defined changes in the composition of small pulmonary arteries. The pulmonary veins also show a certain degree of thickness and a tendency towards arteriolarization. Moreover, in Cpc-PH, the venous system becomes fully arteriolarized, and small arteries exhibit a clear muscularization process and remodeling; an impairment of gas exchange diffusion or a lengthening of the path between air and the red blood cells is prominent.
In an effort to differentiate Cpc-PH from Ipc-PH in a non-invasive manner, a physiologybased approach is important to detect these pathological changes. In our cohort, the low value of peak effort PETCO 2 value, which is one of the ventilatory variables in the CPET, was an optimal indicator of Cpc-PH; thus, it reflected a marked impairment of CO 2 release into the alveolar space.

Usefulness of peak effort PETCO 2 in the detection of Cpc-PH
The differentiation of Cpc-PH between PAH (pre-capillary PH) and PH-LHD (post-capillary PH) using the CPET was reported by Caravita et al. [17]. They found that VE/VCO 2 at the AT was useful in the detection of Cpc-PH and that Cpc-PH was an intermediate between PAH and Ipc-PH in terms of gas exchange. Among the ventilatory parameters obtained using the submaximal exercise test, a low PETCO 2 , high VE/VCO 2 , and high VD/VT were reportedly characteristic of Cpc-PH [16]. Moreover, the exacerbation of pulmonary gas exchange abnormalities in patients with Cpc-PH was related to an excessive rise in the pulmonary vascular pressures [16]. Zhong et al. also reported that VE/VCO 2 -related parameters were diagnostic variables for the presence of pre-capillary components in patients with PH-LHD [24]. Among the ventilatory variables, the lowest VE/VCO 2 %pred, which was obtained from the submaximal exercise test, was the optimal predictor of Cpc-PH (as demonstrated by an AUC of 0.77). From our data, the peak PETCO 2 was also particularly useful in detecting Cpc-PH (as demonstrated by an AUC of 0.73). This is comparable to the findings reported by Zhong et al. In the maximal exercise test, the peak PETCO 2 was the optimal diagnostic variable. Our findings  (i.e., lower peak PETCO 2 and lower ΔVO 2 /ΔWR in CpC-PH) solidified the usefulness of CPET parameters for Cpc-PH detection.
In the HF population, the PETCO 2 is a known CPET variable that potentially possesses prognostic information [25]. In particular, Arena et al. reported that PETCO 2 changes from rest to the RC point, PETCO 2 at the RC point, and PETCO 2 at peak exercise were all significant predictors of cardiac related events. Low value of PETCO 2 during exercise have been classically considered to strongly reflect a low CO during exercise. Matsumoto et al. found that PETCO 2 at the RC point was significantly correlated with the CO at peak exercise in patients with cardiac disease [26]. Furthermore, they concluded that the decrease in CO lead to a decrease in alveolar capillary perfusion and PCO 2 into the alveolar air, resulting in a decrease in PETCO 2 during exercise. Moreover, Tanabe et al. revealed a significant correlation between the PETCO 2 and cardiac index at peak exercise in patients with HF [27].

Mechanisms by which peak effort PETCO 2 predicts Cpc-PH
In patients with PAH, the PETCO 2 decline associated with exercise was more distinct than that in those with LHD [28]. Hemnes et al. demonstrated that the measurement of resting PETCO 2 at bedside may distinguish patients with PAH from those with pulmonary venous hypertension or no PH [29]. Moreover, Welch et al. also demonstrated that this readily available resting PETCO 2 may be a physiologically relevant marker of poor prognosis in PAH [30]. They reported that lung diffusion for carbon monoxide (DLCO) is correlated with the resting PETCO 2 , suggesting that these variables could provide potentially similar insights into the degree of pulmonary vasculopathy in patients with PAH. DLCO measures the ability of a gas to diffuse from the alveoli to the red blood cells in the pulmonary capillaries and depends on the alveolar-capillary membrane diffusive capacity and the capillary volume (which is the amount of blood flowing through the ventilated alveolar-capillary units over a period of time i.e. a few seconds) [31]. Lack of capillary perfusion leads to decrease exchanging gas into the alveolar capillaries and causes increased dead space. The correlation between the PETCO 2 and DLCO suggests that both are markers of dead-space ventilation and that the PETCO 2 may also reflect the capillary membrane diffusive capacity and capillary volume i.e. the CO. The peak PETCO 2 may better capture the pathological changes in Cpc-PH, whereby the venous system becomes fully arteriolarized and gas exchange is strongly impaired.

Usefulness of ΔVO 2 /ΔWR in the detection of Cpc-PH
The lower ΔVO 2 /ΔWR represents a poor increase in CO during exercise (a prognostic predictor), and has been suggested to be related to the severity of HF and pulmonary vascular disease in patients with HF [9,32,33]. Bandera et al. reported that ΔVO 2 /ΔWR flattening was an indicator of an abnormal pulmonary vascular response to exercise (i.e., right ventricular-pulmonary artery uncoupling) [34]. Consistent with these physiological findings, ΔVO 2 /ΔWR was also predictive of Cpc-PH in the present study; a lower ΔVO 2 /ΔWR may represent both a poor increase in CO during exercise and exacerbation of the pulmonary vascular dysfunction in Cpc-PH.

Limitations
This study has certain limitations. First, our study population only included patients who were able to undergo the exercise stress test. Second, owing to the limited sample size, the interpretation of the multivariate analysis results was limited and the statistical tests were underpowered in terms of establishing novel models including several variables to predict Cpc-PH, and further studies with large study populations are warranted. Finally, our cohort comprised a heterogeneous population of patients with cardiac disease.

Conclusions
Our exploratory study reveals that the CPET variables, especially in the lower peak PETCO 2 and lower ΔVO 2 /ΔWR, are useful in differentiating Cpc-PH in patients with LHD. These findings might be explained, at least in part, by the presence and extent of pathologic and physiologic pulmonary vascular changes.